Bone morphogenic protein binding peptide

ABSTRACT

A cyclized peptide designated BMP Binding Peptide (BBP) is a synthetic peptide that avidly binds rhBMP-2, as do endogenous forms of BBP, and sequence conservation between species results in a variety of useful BBP compositions. BBP increases the over-all osteogenic activity of rhBMP-2, increases the rate at which rhBMP-2 induces bone formation, and BBP induces calcification alone. Compositions and substrates including BBP, and methods of using BBP are useful in therapeutic, diagnostic and clinical applications requiring calcification and osteogenesis.

PRIORITY CLAIM

This application is a continuation-in-part of U.S. application Ser. No.10/587,313, which was filed on Jul. 26, 2006, which is a national phaseapplication of PCT Application No. PCT/US2005/002722, filed Jan. 28,2005, which claims priority from U.S. Provisional Patent Application60/539,903 filed Jan. 28, 2004.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 20, 2010, isnamed 38586357.txt and is 11,264 bytes in size.

BACKGROUND OF THE INVENTION

Growth factors are substances, including peptides, which affect thegrowth and differentiation of defined cell populations in vivo or invitro. Normal bone formation occurs during development, bone remodelingoccurs in adult life, and bone repair occurs in order to preserve theintegrity of the skeleton. Bone formation, remodeling and repair involvebone resorption by osteoclasts and bone formation by osteoblasts. Celldifferentiation and the activity of osteoblasts and osteoclasts areregulated by growth factors. Thus, any interference between the balancein cell differentiation and resorption can affect bone homeostasis, boneformation and repair.

Osteoblasts are derived from a pool of marrow stromal cells (also knownas mesenchymal stem cells). MSC are present in a variety of tissues andare prevalent in bone marrow stroma. MSC are pluripotent and candifferentiate into chondrogenic or osteogenic cells includingosteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes.

The induction of ectopic bone formation by demineralized bone matrix(DBM) has been described. (Urist, M. R.: Bone: Formation byautoinduction. Science 150:893-899, 1965; Urist, et al., Purification ofbovine morphogenetic protein by hydroxyapatite chromatography. Proc.Natl. Acad. Sci. USA 81:371-375, 1984; Urist, M. R. Emerging concepts ofbone morphogenetic protein. In Fundamentals of Bone Growth: Methodologyand Applications, Boston C.R.C. Press, pp. 189-198, 1991.) Further, theproperties of the partially purified protein fraction, bone morphogenicprotein/non-collagenous protein (“BMP/NCP” or “BMP”s) have beendescribed. (Urist, et al. Methods of Preparation and Bioassay of BoneMorphogenetic Protein and Polypeptide Fragments. In Methods inEnzymology. Vol. 146. New York, Academic Press, pp. 294-312, 1987;Urist, et al., Hydroxyapatite affinity, electroelution, andradioimmunoassay for identification of human and bovine bonemorphogenetic proteins and polypeptides. In Development and Diseases ofCartilage and Bone Matrix. New York, Alan R, Liss, Inc., pp. 149-176,1987.)

BMP/NCP was never purified to homogeneity, but other investigators haveused similar starting materials to clone a number of recombinant “BMPs.”However several of these molecules have little or no osteogenicactivity. “BMPs” and other osteogenic factors have been studied for usein clinical applications. However, the cost of using minimally effectivedosages of BMP-7, for example has been a limiting factor in clinicaluse. Therefore, effective and affordable compositions and methods aredesired for clinical applications relating to bone.

BRIEF SUMMARY OF INVENTION

The inventions are related to a synthetic peptide designated BMP BindingPeptide (BBP) that avidly binds rhBMP-2. BBP increases the rate anddegree to which rhBMP-2 induces bone formation. BBP alone inducescalcification of chondrogenic, osteogenic and osteoblastic cells.Compositions and substrates including BBP, antibodies to BBP and methodsof using BBP are useful in applications relating to bone.

The invention may include a method of systemic delivery or localizedtreatment with agents for maintaining bone homeostasis, enhancing boneformation and/or enhancing bone repair.

In one application of the invention, the method may be applied to inducethe local repair of bone or to treat bone related disorders, such asosteoporosis.

The invention may also include implants having agents or seeded withpluripotential or differentiated cells for inducing bone formation orrepair.

The invention may also include the application of substances ordifferentiated cells at a site where bone formation or bone repair isdesired.

This invention is advantageous at least in that BBP enhancescalcification of chondrogenic or osteogenic precursor cells. Further,this invention is advantageous at least in that BBP enhancesosteogenesis to occur faster to a greater extent, which may improve theclinical rate and effectiveness of treatment with BMP, and reduce dosesand therefore the cost of treatment.

These, as well as other objects, features and benefits will now becomeclear from a review of the following detailed description ofillustrative embodiments and the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A are BBP bovine (1) amino acid and (2) nucleic acid sequences,respectively; FIG. 1B is a partial amino acid sequence of the bovine BMPbinding protein (“BBP”) showing the cystatin homology region, the BMP-2homology region, and the TGF-β receptor II homology domain.

FIG. 2 is an amino acid sequence alignment of human BMP-2 and the BMP-2homology region in bovine SPP-24; (i, identical; c, conservativesubstitution; sc, semi-conservative substitution).

FIG. 3 is an amino acid sequence alignment of bovine fetuin and humanTGF-β receptor II (above) and of human TGF-β receptor II and the TGF-βreceptor II homology domain of bovine SPP-24 (corresponding to BBP)(bottom); (i, identical; c, conservative substitution; sc,semi-conservative substitution).

FIG. 4 is a radiogram of mouse hind quarters 21 days after implantationof 500 μg of BBP in atelocollagen (top) or atelocollagen alone (bottom).

FIG. 5 is a histological section of mouse muscle 21 days afterimplantation of 500 μg of BBP in atelocollagen. (H & E stain. Originalmagnification 100×.)

FIG. 6 are radiograms of mouse hind quarters 21 days after implantationof 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2 plus 500 mg of BBP (right).

FIG. 7 are radiograms of mouse hind quarters 9 (top) and 12 (bottom)days after implantation of 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2plus 500 mg of BBP (right).

FIG. 8 are histological sections of mouse hind quarters 9 days afterimplantation of 5 μg of rhBMP-2 alone (A) or 5 μg of rhBMP-2 plus 500 μgof BBP (B).

FIG. 9 is a surface plasmon resonance sensogram for the interaction ofrhBMP-2 (affixed to the chip) and cyclized BBP at concentrations rangingfrom 1×10⁻⁵ M 1×10⁻⁴ M.

FIG. 10 is a bar graph depicting the percentage of rhBMP-2 retentionover 1, 3 and 7 days in the presence or absence of BBP.

FIG. 11 includes amino acid sequences against which specific SSP-24/BBPantibodies have been generated.

FIGS. 12 A & B depict flowcharts of exemplary methods of the invention.

FIGS. 13 A & B are schematic depictions of two embodiments of theinvention.

FIG. 14 A is a chart showing the amino acid sequences for BPP in variousspecies (SEQ ID NOS 11, 1, 13, 15, 17, 19, 21, 23, 25 & 27 are disclosedrespectively in order of appearance). FIG. 14 B is a list of the nucleicacid sequences for BPP in various species (SEQ ID NOS 12, 14, 16, 18,20, 22, 24, 26 & 28 are disclosed respectively in order of appearance).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

One embodiment of the invention comprises a peptide having the aminoacid sequence of SEQ ID NO: 1. The bovine derived amino acid SEQ ID NO:1 has been designated BBP, and SEQ ID NO: 2 corresponds to the bovinenucleic acid sequence encoding BBP.

One embodiment of the invention comprises a peptide having the aminoacid sequence of SEQ ID NO: 13, which is the sequence of human BBP. SEQID NO: 14 corresponds to the human nucleic acid sequence encoding humanBBP.

BBP is a 19 amino acid, 2.1 kD peptide, derived from a 18.5 kD fragmentof a known 24 kDa secreted phosphoprotein (“SPP-24”). SPP-24 isillustrated in FIG. 2. Notably, SPP-24 inhibits BMP-2 induced boneformation. BBP contains the cystatin-like domain of SPP-24. BBP isexpressed at least in the liver and bone (including at leastdemineralized cortical bone and periosteum).

The BBP amino acid sequence is similar to the TGF-β/BMP-binding regionof fetuin, a member of the cystatin family of protease inhibitors. BBPavidly binds rhBMP-2 (recombinant human BMP-2) with a K_(D) of ×10⁻⁵ M.BBP may also bind other molecules having similar binding domains toBMP-2, such as other TGF-β proteins (including but not limited to BMP-4and BMP-7) and affect their retention rates and/or activity as well.

BBP alone induces calcification of vertebrate chondrogenic andosteogenic precursor cells. BBP increases the increases the rate anddegree to which rhBMP-2 induces bone formation. Surprisingly, BBP asused with BMP-2 in vivo causes osteogenesis to occur faster and to agreater extent and with smaller amounts of rhBMP-2.

For example, when implanted alone in mouse muscle, the BBP inducesdystrophic calcification. The process of bone formation in repair orectopic bone formation the “mouse hindquarter” or “muscle pouch” modelrecapitulates endochondral bone formation. The first step involves theproduction of cartilage, which is replaced by bone. This same processthat occurs during endochondral bone formation in development, whilesome membraneous bone formation occurs directly without a cartilageintermediary.

In one embodiment of the invention, a peptide comprising a fragment ofBBP may be useful, if the fragment similarly increases degree or rate ofosteogenesis by BMP-2 in mammalian cells, or increases degree or rate ofcalcification in vertebrate cells, or specifically mammalianchondrogenic or osteogenic progenitor cells.

Forms of BBP having modifications of the amino acid SEQ. ID NO: 1 mayalso be useful in this invention. For example, the conserved amino acidsequences of BBP between species, deletional or insertionalmodifications, conservative or semi-conservative substitutionalmodifications are intended to be encompassed in the claimed BBP, to theextent that the modified amino acid sequences increase the residencytime and or activity of BMP-2 or other TGF-β homologous molecules. BBPis a β-pleated sheet-turn-beat pleated sheet molecular motif (“B-T-B”).It is currently believed that growth factor binding amino acids residein the T-section. Therefore, amino acid substitutions in the T-sectionmay affect activity of BBP to a greater extent than substitutions in theB regions.

One embodiment of the invention comprises a peptide having the sequenceof SEQ ID No. 11: C-R-S-T-V-X-Y-S-X-X-X-V-X-X-V X-Y-Y-C, which is themammalian consensus sequence for BBP. FIG. 14A shows the homology inamino acid sequence across bovine (SEQ ID No. 1; nucleic acid sequenceset forth at SEQ ID No. 2), human (SEQ ID No. 13; nucleic acid sequenceset forth at SEQ ID No. 14 (position 9 is either A or V), porcine (SEQID No. 15; nucleic acid sequence set forth at SEQ ID No. 16), ovine (SEQID No. 17; nucleic acid sequence set forth at SEQ ID No. 18), rat (SEQID No. 19; nucleic acid sequence set forth at SEQ ID No. 20), and mouse(SEQ ID No. 21; nucleic acid sequence set forth at SEQ ID No. 22) BBP.FIG. 14A also shows highly conserved regions in chicken (SEQ ID No. 23;nucleic acid sequence set forth at SEQ ID No. 24), salmon (SEQ ID No.25; nucleic acid sequence set forth at SEQ ID No. 26) and trout (SEQ IDNo. 27; nucleic acid sequence set forth at SEQ ID No. 28).

In FIG. 14 A, “X” and “Z” are used to denote amino acid substitutionsthat are understood to be semi-conservative or conservative,respectively. Conservative substitutions include amino acids selectedfrom the same group, and semi-conservative substitutions includesubstitutions that are not believed to affect the BMP-2 binding domainor the function of the BBP. For example, the substitution at position 6is conservative between human, rat and ovine, but semi-conservative withsome other species because the amino acids reported at that position indifferent species are: Q and E (Q in porcine, rat, and mouse BBP, and Ein chicken). Although K and R are both classified as basic amino acids,Q is classified as an uncharged polar amino acid, therefore thesubstitution is not conservative. The substitution is semi-conservative,however, because the function of BBP is believed to be unaffected.Semi-conservative substitutions are also found at positions 9, 10, 11,and 16. At position 9, the amino acids A is found in bovine, human,porcine and ovine BBP, compared to K in rat and mouse BBP. At position10, the amino acid E is reported for bovine, porcine, and ovine BBP,human BBP contains Q at that position, and rat and mouse BBP contain theamino acid G. At position 11, the amino acid Q is found in bovine,human, rat and mouse, whereas K, is reported for porcine BBP and R forovine BBP. At position 16, W is found in bovine, porcine, ovine, rat andmouse BBP, whereas human BBP contains an H. There are alsosemi-conservative substitutions at positions 13 and 14 betweenrat/human, as opposed to other species.

An example of a conservative substation is found at position 7. At thisposition, different hydrophobic amino acids are observed in differentspecies, namely, M in bovine, ovine, rat, and mouse BBP, compared to Vin human and I in porcine BBP. This substitution is consideredconservative because M, V, and I are all hydrophobic amino acids. Otherconservative substitutions occur at positions 17 and 18. Two hydrophobicamino acids, A and V, are found at position 17. At position 18, twobasic amino acids, R and H, are found.

One embodiment of the invention may be a composition including BBP whichincreases degree or rate of calcification in vertebrate cells, or morespecifically mammalian chondrogenic or osteogenic precursor cells.Further, the invention may be including BBP which increases degree orrate of osteogenesis by BMP-2, and one of BMP-2 or demineralized bonematrix. Further, the composition may additionally or alternativelyinclude other TGF-13 family members, including but not limited to BMP-4or BMP-7. It is further noted that other TGF-13 family members areinvolved in immune system function, and BBP may bind with an effect theresidence time or activity of those molecules, as well which may effectimmune function, inflammation or tumor growth.

In one embodiment, the invention may include a medicament for use ininducing the rate or degree of osteogenesis in a vertebrate including atherapeutically effective dosage of BBP and BMP or DBM. The inventionmay further include, a medicament for use in inducing the rate or degreeof calcification in a vertebrate including a peptide comprising BBP.

Applications for BBP. A number of applications for BBP are suggestedfrom its pharmacological (biological activity) properties. For example,BBP alone or in combination with other TGF-family members such as BMP-2,BMP-4 and BMP-7, or demineralized bone matrix may be used in clinical orresearch methods for inducing bone formation, maintaining bonehomeostasis and/or enhancing bone repair. BBP may be used alone or incombination to treat developmental or homeostatic bone disorders (suchas osteoporosis), bone injury (such as fracture healing flat (e.g.,membranous) and long (e.g., endochondral) bones [comment that this isequally applicable], non-union fractures and reconstructive surgery. Theinvention may also be used in treating periodontitis, periodontalregeneration, alveolar ridge augmentation for tooth implantreconstruction, treatment of non-union fractures, sites ofknee/hip/joint repair or replacement surgery.

Clinical indices of a method or compounds ability to maintain bonehomeostasis is evidenced by improvements in bone density at differentsites through out the body as assessed, at least by DEXA scanning.Enhanced bone formation in a healing fracture is routinely assessed byregular X-ray of the fracture site at selected time intervals. Moreadvanced techniques for determining the above indices, such asquantitative CT scanning or quantitative histological methods (eg.,tissue is processed, stained, and microscopically examined and bonedefined an measured with image analysis) may be used. Further, measuresof bone density, bone area, bone mineral content, formation of ectopicbone, and increases in the opacity of tissue upon X-ray examination,expression of alkaline phosphatase activity, calcium incorporation,mineralization or expression of osteocalcin mRNA may be used to observethe effects of BBP calcification and/or osteogenesis

The invention may also include the use of agents which inhibitosteoclastic bone resorption. Agents which may be useful in thisinvention to effect osteoclastic bone resorption include, but are notlimited to, bisphosphonates, the selective estrogen receptor modulators,calcitonin, and vitamin D/calcium supplementation. The invention mayalso include the use of agents which induce osteoblastic bone formation.Agents which may be useful in this invention include, but are notlimited to PTH, sodium fluoride and growth factors, such as insulin-likegrowth factors I and II.

The in vivo models used to show the calcification effects of BBP aloneor osteogenic effects in combination with BMP have been used previouslyin demonstrating similar behaviors of other compounds. In particular, invivo models have also previously been able to successfully predict thein vivo osteogenic effects of compounds such as BMP and insulin likegrowth factors (IGF). Specifically, it has been demonstrated that theosteogenic effects of BBP in an animal model using rat femur, ectopicbone formation model. Therefore it is anticipated that, based on thesesimilar findings, BBP will have osteogenic effects in vivo in humans.Demonstration of osteogenic effects of a compound in these in vivomodels are necessary prior to trials that would demonstrate theireffects in vivo humans.

Therapeutically effective dose. A therapeutically effective dose of BBPor a TGF-β family member useful in this invention is one which has apositive clinical effect on a patient or desired effect in cells asmeasured by the ability of the agent to enhance calcification orosteogenesis, as described above. The therapeutically effective dose ofeach agent can be modulated to achieve the desired clinical effect,while minimizing negative side effects. The dosage of the agent may beselected for an individual patient depending upon the route ofadministration, severity of the disease, age and weight of the patient,other medications the patient is taking and other factors normallyconsidered by an attending physician, when determining an individualregimen and dose level appropriate for a particular patient.

This invention is advantageous in at least the dosage of BMP-2 requiredto induce a given rate or degree of osteogenesis may be reduced whenBMP-2 is combined with BBP.

Dosage Form. The therapeutically effective dose of an agent included inthe dosage form may be selected by considering the type of agentselected and the route of administration. The dosage form may include aagent in combination with other inert ingredients, including adjutantsand pharmaceutically acceptable carriers for the facilitation of dosageto the patient, as is known to those skilled in the pharmaceutical arts.

Therapeutic formulations of BBP (when claimed is intended to includemodifications or fragments thereof), may be prepared for storage bymixing the BBP having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers, in theform of lyophilized cake or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; anti-oxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins. Other components caninclude glycine, blutamine, asparagine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics orpoly(ethylene glycol) (PEG).

The dosage form may be provided in preparations for subcutaneous (suchas in a slow-release capsule), intravenous, intraparitoneal,intramuscular, peri- or intraskeletal for example. Any one or acombination of agents may be included in a dosage form. Alternatively, acombination of agents may be administered to a patient in separatedosage forms. A combination of agents may be administered concurrent intime such that the patient is exposed to at least two agents fortreatment.

Additional Agents. The invention may include treatment with anadditional agent which acts independently or synergistically with BBP toenhance calcification osteogenesis. For example, BBP may be combinedwith BMP, bisphosphonates, hormone therapy treatments, such as estrogenreceptor modulators, calcitonin, and vitamin D/calcium supplementation,PTH (such as Forteo or teriparatide, Eli Lilly, sodium fluoride andgrowth factors that have a positive effect on bone, such as insulin-likegrowth factors I and II and TGF-β. Those skilled in the art would beable to determine the accepted dosages for each of the therapies usingstandard therapeutic dosage parameters, or reduced dosages where theeffects of BBP are synergistic with the secondary agent, such as BMPs.

BBP is currently thought to act upon BMP-2 at least by increasing itsresidency time with a substrate. One embodiment of the invention is amethod of detecting the ability of BBP to enhance the residency time ofa TGF-β homologous molecule including applying an amount of the TGF-βhomologous molecule at a first and second selected location. Further,applying a selected amount of BBP at the first selected location, andfinally detecting the amount of the TGF-β homologous molecule at thefirst and second location after a selected time period; and calculatingthe difference between the amount of the TGF-β homologous molecule atthe first and second location.

In one embodiment, the invention may include a method of enhancing therate or degree of osteogenesis in vertebrate tissue includingapplication of BBP which increases degree or rate of osteogenesis byBMP-2 in mammalian cells and one of a TGF-β family member, such as BMP-2or demineralized bone matrix.

In one embodiment, the invention may include a method of inducingcalcification of vertebrate tissue, or more specifically vertebratechondrogenic or osteogenic precursor cells, including application ofBBP.

In one embodiment, the invention may include a method of enhancing therate or degree of osteogenesis in vertebrate tissue includingadministering chondrogenic or osteogenic precursor cells to the patientat a location proximate to the desired location of osteogenesis;further, administering BBP, and administering one of a TGF-β familymember, such as BMP-2 or demineralized bone matrix.

In one embodiment, the invention may include a method of enhancing therate or degree of calcification in vertebrate tissue includingadministering osteogenic cells to the patient at a location proximate tothe desired location of calcification and further, administering BBP.

In one embodiment, the invention may include method of enhancing therate or degree of osteogenesis in a vertebrate including treatingvertebrate mesynchymal stem cells with one of a TGF-β family member,such as BMP-2 or demineralized bone matrix to induce osteogenesis of thecells. Further, treating the vertebrate mesynchymal stem cells with BBP;and administering the vertebrate mesynchymal stem cells to the patientat a location proximate to the desired location of osteogenesis.

For example, mammalian cells, such as mesenchymal stem cells can beharvested, from the patient or a cell donor. The cells may be injectedin a location where bone formation or repair is desired (such as afracture site or implant site), or first treated with BBP and/or BMP.The cells may then be re-administered to the patient, eithersystemically or at a selected site at which osteogenesis ofcalcification is desired. Additionally, the patient may by treatedlocally or systemically with at least one additional agent which effectsosteogenesis or calcification.

FIGS. 12A and B depict flowcharts of exemplary methods of the invention,the steps of which may be performed in any order.

One embodiment of the invention may include an article of manufacturecomprising BBP immobilized on a solid support. The solid support mayfurther include a TGF-β family member, such as BMP-2 or demineralizedbone matrix.

One embodiment of the invention may include an implant for use in vivoincluding, a substrate where at least the surface of the implantincludes BBP. The implant may further include MSC, chondrocytic orosteoblastic progenitor cells. Further, the implant may be formed intothe shape of a pin, screw, plate, or prosthetic joint, for example.

For example, FIGS. 13A & B depict two embodiments of the presentinvention. In FIG. 13A, the invention may include implants or grafts(200) for use in the body comprising, a substrate having a surface(201), wherein at least the surface of the implant includes BBP (203) inan amount sufficient to induce, calcification or osteogenesis in thesurrounding tissue. The implant may include mesynchymal stem cell,chondrogenic or osteogenic cells expressing BBP, and/or BMP-2,demineralized bone matrix, or collagen cultures. The implant may be inthe form of, but are not limited to pins, screws, plates or prostheticjoints which may be placed in the proximity of or in contact with a bone(202) that are used to immobilize a fracture, enhance bone formation, orstabilize a prosthetic implant by stimulating formation or repair of asite of bone removal, fracture or other bone injury (204).

As shown in FIG. 13B, the invention may also include the in vitro (suchas on cultures of collagen or chondrocytes) or in vivo application of ata least BBP containing composition or BBP expressing cells (206) in theproximity of or in contact with a bone (202), an implant (200) at a siteof bone removal, fracture or other bone injury (204) where osteogenesisand/or calcification is desired. The BBP composition may be applied incombination with other agents such as BMP-2, demineralized bone matrix,or collagen cultures.

For example, the use of stem cells for treating bone related disordersin humans has also been examined. Infusion of osteoblastic progenitorstem cells from a healthy individual into a diseased individual has beenshown to improve bone density in these patients (OI). Cells may bepretreated with BMP and BPP, or applied concurrently therewith.

In one embodiment, the invention may include a monoclonal or polyclonalantibody having selective binding to any portion of BBP, or the BBPportion of the BBP precursor, SSP-24.

BBP or fragments thereof may be fused (for example by recombinantexpression or in vitro covalent methods) to an immunogenic polypeptideand this, in turn, may be used to immunize an animal in order to raiseantibodies against BBP. Antibodies are recoverable from the serum ofimmunized animals. Alternatively, monoclonal antibodies may be preparedfrom cells from the immunized animal in conventional fashion.Immobilized antibodies may be useful particularly in the detection orpurification of BBP.

Two examples of specific peptide sequences against which rabbitpolyclonal antibodies have been generated include: (1) An antibodyagainst the peptide sequence “IQETTCRRESEADPATCDFQRGYHVPVAVCRSTVRMSAEQV”(FIG. 11—SEQ. ID NO: 3) that reacts with both bovine and human SSP-24,the BBP precursor. This antibody was generated in rabbits immunized withthe synthetic peptide indicated above. Further, (2) An antibody directedagainst the sequence “CGEPLYEPSREMRRN” (FIG. 11-SEQ. ID NO: 4) that wasalso produced in rabbits immunized with a synthetic peptidecorresponding to the indicated sequence. This second antibody reactswith bovine SSP-24. The N-terminal cysteine is not a part of the nativeSSP-24 sequence; but is preferably included to allow the peptide to beconjugated to chromatographic resins for affinity chromatography.Additional peptide sequences may be identified for specific binding toBBP, and sequences may be selected so as to create an antibody havingselective binding with BBP, but so as to not interfere with BBP binding,such as the region of BBP which binds with BMP-2 or other TGF-β familymembers.

Antibodies against the sequences above, corresponding sequences in themouse, human, and rat genome, or any derivatives of the immunogenicsequences are also useful in this invention. These antibodies are usefulin at least to the extent that they recognize the BBP amino acidsequence with high specificity. Such antibodies may also be useful ininhibiting protein specific interactions of BBP with other moleculeswhere the antibody binds to a location on the peptide which interactswith other molecules. The inhibition of BBP activity in situations wherethe rate or degree of chondogenesis or osteogenesis may be modified.

In one embodiment the invention, antibodies specific for BBP may beuseful in decreasing the degree or rate of osteogenesis by BMP-2 invertebrate cells or decreasing degree or rate of calcification invertebrate cells, or more specifically in mammalian chondrogenic orosteoblastic precursor cells.

One embodiment of the invention may also include a method of using BBPselective antibodies to detect the presence of SSP-24/BBP in sample(including but not limited to a cell culture, tissue sample, peptidefraction, Western blot) including exposing the sample to the BBPselective antibody and visualizing the complex of SSP-24/BBP and BBPantibody.

In one embodiment of the invention, BBP antibodies may be used for theaffinity purification of the BBP from recombinant cell culture ornatural sources. BBP antibodies that do not detectably cross-react withother growth factors can be used to purify BBP from these other familymembers.

In one embodiment, the invention may include a nucleic acid constructcomprising a DNA or RNA nucleic acid sequence encoding BBP, or modifiedsequences corresponding to the modified amino sequences described above.

The invention may also include, an expression vector operatively linkedto a nucleic acid sequence encoding BBP, or precursor SSP-24 Further, atransformant may be obtained by introducing the nucleic acid constructencoding for BBP, or its precursor SSP-24 into a host cell.

Practice of this invention may include the use of an oligonucleotideconstruct comprising a sequence coding for BBP and for a promotersequence operatively linked in a mammalian or a viral expression vector.Expression and cloning vectors contain a nucleotide sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchcloning vectors are well known to those of skill in the art. Expressionvectors, unlike cloning vectors, may contain an inducible orconstitutive promoter which is recognized by the host organism and isoperably linked to the BBP nucleic acid. The nucleic acid may beoperably linked when it is placed into a functional relationship withanother nucleic acid sequence. For example, DNA for a pre-sequence orsecretory leader is operably linked to DNA for a polypeptide if it isexpressed as a pre-protein which participates in the secretion of thepolypeptide.

One embodiment of the invention may also include a method of using DNAor RNA nucleic acid sequences complimentary and having specific bindingfor the DNA or RNA sequences encoding BBP to detect the presence of BBPDNA or RNA in a sample, respectively (including but not limited to acell culture, tissue sample, nucleic acid fraction, or Southern orNorthern blot) including exposing the sample to the complimentary BBPDNA or RNA sequences and visualizing the complex of hybrids.

Example 1 Extraction and Separation of Non-Collagenous Bone Proteins(NPCs)

Methods: NCPs were extracted from defatted, demineralized human corticalbone powder with 4 M GuHCl, 0.5 M CaC1₂, 2 mM N-ethylmalemide, 0.1 mMbenzamidine HCl, and 2 mM NaN₃ for 18 hr at 6° C. Residual collagen andcitrate-soluble NCPs were extracted by dialysis against 250 mM citrate,pH 3.1 for 24 hours at 6° C. The residue was pelleted by centrifugation(10,000×g at 6° C. for 30 min), defatted with 1:1 (v/v) chloroform:methanol for 24 hr at 23° C., collected by filtration and dried at 22°C. The material was resuspended in 4 M GuHCl, dialyzed against 4 MGuHCl, 0.2% (v/v) Triton X-100, 100 mM Tris-HCI, pH 7.2 for 24 hr at 6°C., then dialyzed against water, and centrifuged at 10,000×g for 30 minat 6° C. The pellet was lyophilized and subsequently separated byhydroxyapatite chromatorgraphy.

Chromatography was conducted using a BioLogic chromatography workstationwith a CHT-10 ceramic hydroxyapatite column (BioRad, Hercules, Calif.).Bovine BMP/NCP was solublized in 6 M urea, 10 mM sodium phosphate, pH7.4. The sample was loaded onto the hydroxyapatite column and theunbound fraction was collected. Bound proteins were eluted withincreasing concentration of sodium phosphate to 300 mM over a lineargradient of five column volumes. Five ml fractions were collected duringthe course of the run. The fraction which separated at 180 mM phosphatewas separated further by SDS-PAGE electrophoresis. A band correspondingto a M_(r) of 18.5 was excised and submitted for sequence analysis bymatrix assisted laser-desorption ionization/time of flight massspectroscopy (MALDI/TOF MS).

Results: Sequence Identification and Analysis: The fraction of bBM P/NCPwhich eluted from hydroxyapatite at 180 mM phosphate was separated bySDS-PAGE electrophoresis and the material with a M_(r) of 18.5 kD wassubmitted for MALDI/TOF MS analysis. The major protein component of thismaterial was determined to be a fragment of SPP-24 on the basis of sixpeptides with sequences identical to regions of that protein. (Hu, etal., Isolation and molecular cloning of a novel bone phosphoproteinrelated in sequence to the cystatin family of thiol protease inhibitors.J. Biol. Chem. 270:431-436, 1995.) The sequences of these peptides areshown in Table 1.

TABLE 1 Identification of the 18.5 kD protein by MALDI/TOF massspectroscopy and peptide fingerprinting. Expected Mass^(a) ObservedMass^(a) Peptide Sequence 1526.574 1526.53 ESEADPATCDFQR* 1411.6001411.71 VNSQSLSPYLFR 1291.406 1291.41 SRGEPLYEPSR 1249.409 1249.48NSYLLGLTPDR 1158.363 1158.27 GYHVPVAVCR* *modified cystein; ^(a)=peptide masses are expressed as [M + H⁺]

Analysis of this sequence with the SWISS-PROT data base revealed thecystatin-like domain which had been previously described, but no othersequence similarities of relevance to bone metabolism. (Hu, et al.)However, it is known from other work that other cystatin-like proteinsinteract with proteins having a role in bone metabolism. Specially,members of the cystatin family have TGF-β and BMP-2 binding propertiesbased on similarities to the TGF-β receptor. (Brown, et al., Friends andrelations of the cystatin superfamily—new members and their evolution.:Protein Sci. 6:5-12, 1997; Demetriou, et al., Fetuin/a2-HS glycoproteinis a transforming growth factor-(3 type II receptor mimic and cytokineantagonist. J. Biol. Chem. 271:12755-12761, 1996.) However, fetuinantagonizes BMP activity. (Hu, et al.) Therefore, a manual comparisonwas made of the cystatin-like region of SPP-24 and the cystatin-likedomain of fetuin.

FIG. 1B is a partial amino acid sequence of the bovine SSP-24, the BMP-2homology region, and the TGF-β receptor II homology domain. Underlinedamino acids have been confirmed to be present by mass spectroscopy.(GenBank Accession Number U08018; Hu, et al.)

Two regions of interest were identified in the cystatin-like region ofSPP-24. One region had some sequence similarity to BMP-2, whereas theother region had sequence similarity to the TGF-β receptor II homologydomain of fetuin. That part of the sequence of SPP-24 which containsthese two regions is shown in FIG. 1B.

Comparisons of the two regions of interest to human BMP-2 and humanTGF-β receptor II are shown in FIGS. 2 and 3. FIG. 2 is an amino acidsequence alignment of human BMP-2 and the BMP-2 homology region inbovine SPP-24. FIG. 3 is an amino acid sequence alignment of bovinefetuin and human TGF-β receptor II (top) and of human TGF-β receptor IIand the TGF-β receptor II homology domain of bovine SPP-24(corresponding to BBP) (bottom). Alignment of the SPP-24, fetuin, humanBMP-2, and human TGF-β receptor II sequences was accomplished using theT-Coffee program. (Notredame, et al, T-Coffee: A novel method formultiple sequence alignments. J. Molecular Biol. 302:205-217, 2000.)Synthetic peptides corresponding to these two regions were obtained andsubjected to chemical and in vivo analysis as described below.

Example 2 In Vivo Activity of BBP

Methods: The osteogenic activity of material was tested using maleSwiss-Weber mice aged 8 to 10 weeks were used (Taconic Farms,Germantown, N.Y.). Prior to the assay, the BBP was solublized andlyophilized into 2 mg of atelocollagen. The dried material was placed ina #5 gelatin capsule and sterilized by exposure to chloroform vapor. Toconduct the assay, mice were anesthetized using 1% isoflurane deliveredin oxygen at 2 l/min through a small animal anesthesia machine(VetEquip, Pleasanton, Calif.). Animals were affixed to a surgery boardand the fur over the hindquarters shaved. The skin was cleaned with 70%ethanol and a midline incision made over the spine adjacent to thehindquarters. Blunt dissection with scissors was used to expose thequadriceps muscle on one side. A small pouch was made in the muscleusing the point of scissors and the #5 capsule containing the testmaterial was inserted into the pouch. The skin was then closed withthree 11 mm Michel surgical clips and the animal returned to its cagefor monitoring.

After 21 days the animals were killed and the hindquarter removed.Radiological examination of the specimens was accomplished using a smallparts X-Ray cabinet (Faxitron, Wheeling, Ill.). For quantization of boneformation, bone area and the bone mineral content (BMC) of an area ofinterest encompassing the site of ectopic bone formation was determinedusing a PIXImus2 small animal densitometer (GE Lunar, Madison, Wis.).Specimens were then placed in buffered formalin and submitted forroutine processing for histological examination.

Various amounts of rhBMP-2 and BBP were combined and prepared forimplantation. All possible combinations of the following amounts wereused in pilot studies, rhBMP-2: 0 μg, 0.05 μg, 0.5 μg, 5 μg, and 50 μg;BBP: 0 μg, 50 μg, and μg 500 mg. Samples of 5 μg of rhBMP-2 were used inmore extensive subsequent studies because that amount consistentlyproduced an amount of ectopic bone that was neither too large nor toosmall for reliable analysis.

Results: BBP was tested alone and in combination with rhBMP-2.

FIG. 4 is a radiogram of mouse hind quarters 21 days after implantationof 500 μg of BBP in atelocollagen (top) or atelocollagen alone (bottom).When implanted alone with carrier, BBP induced calcification.

FIG. 5 is a histological section of mouse muscle 21 days afterimplantation of 500 μg of BBP in atelocollagen. Note the dystrophiccalcification primarily associated with intramuscular adipose tissue. (H& E stain. Original magnification 100×.)

When 500 μg of BBP with sequence similarity to the TGF-β receptor II wasimplanted with 5 μg of rhBMP-2 the amount of ectopic bone formed, asmeasured by densitometry, was consistently greater than the amount ofbone formed in animals into which identical amounts of the rhBMP-2 alonewere implanted.

FIG. 6 are radiograms of mouse hind quarters 21 days after implantationof 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2 plus 500 mg of BBP (right).Note the increased opacity associated with the samples containing bothrhBMP-2 and BBP.

Furthermore, implants that contained both the peptide and rhBMP-2produced detectable cartilage and bone earlier than implants of BMP-2alone.

FIG. 7 are radiograms of mouse hind quarters 9 (above) and 12 (below)days after implantation of 5 μg of rhBMP-2 (left) or 5 μg of rhBMP-2plus 500 mg of BBP (right). Note the appearance of calcification in thesample from the day 9 sample containing both rhBMP-2 and BBP but not thesample containing BMP-2 alone.

FIG. 8 are histological sections of mouse hind quarters 9 days afterimplantation of 5 μg of rhBMP-2 alone (A) or 5 μg of rhBMP-2 plus 500 μgof BBP (B). Note the abundant cartilage in the BMP+BBP specimen whereasthe BMP alone specimen shows the earlier stages of inflammation andmesodermal cell proliferation.

TABLE 2 Densitometric quantitation of ectopic bone formation withvarious amounts of BBP implanted with 5 μg of rhBMP-2. Mean, SE (n). BBP(μg) 0 50 500 Bone Area  0.089 ± 0.0336  0.159 ± 0.0606  0.226 ± 0.0270(cm²) (12)* (8) (12)* Bone 0.00189 ± 0.00084 0.00388 ± 0.0017 0.00528 ±0.00068 Mineral (12)** (8) (12)** Content (g) *p = 0.0044; **p = 0.0049

Example 3 Surface Plasmon Resonance to Determine the Interaction ofBmp-2 and the Synthetic Peptide

Methods: The binding interaction between rhBMP-2 and BBP wascharacterized using surface plasmon resonance employing a Biacom Xinstrument (Biacore, Piscataway, N.J.). Buffers and chips for theprocedure were obtained from Biacore. RhBMP-2 was dialyzed into 10 mMsodium acetate, pH 5.5 at a concentration of 1 mg/ml. This material wasthen attached to a CM-5 sensor chip using reagents and proceduressupplied by the manufacturer. Running buffer was 10 mM HEPES, pH 7.4,150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20. The peptide was dissolvedin running buffer at concentrations ranging from 1×10⁻⁵ to 1×10⁻⁴ M.Flow rates from 5 to 50 μl/min and injection volumes of 20 to 100 μlwere employed. The regeneration solution was 10 μM glycine-HCl, pH 2.0.

Results: Results of the surface plasmon resonance studies to determinethe interaction between rhBMP-2 and BBP are shown in FIG. 9.

FIG. 9 is a surface plasmon resonance sensogram for the interaction ofrhBMP-2 (affixed to the chip) and cyclized BBP at concentrations rangingfrom 1×10⁻⁵ M 1×10⁻⁴ M. The estimated dissociation constant (K_(D)) forthe interaction was 3×10⁻⁵ M. When the BBP was decyclized by priorreduction with β-mercaptoethanol, no significant binding occurred.

Example 4 Residence Time Study: BBP and rhBMP-2

Methods: Labeled rhBMP-2 was mixed with BBP or vehicle and applied tocollagen sponges. The sponges were implanted into muscle pouches inrodents. At specified times (1, 3 and 7 days), the implants were removedand the amount of BMP remaining determined. Four animals were used ineach group.

Results: BBP increased retention of rhBMP-2 by a factor of about two.FIG. 10 is a bar graph depicting the percentage of rhBMP-2 retentionover 1, 3 and 7 days in the presence or absence of BBP.

Discussion: Increasing the retention of BMP at an implant site mayimprove the effectiveness of the BMP, and also reduce the amountrequired for the same therapeutic result.

While the specification describes particular embodiments of the presentinvention, those of ordinary skill can devise variations of the presentinvention without departing from the inventive concept.

Example 5 In Vivo Activity of Human BBP

Methods: The methods of Example 5 were utilized to test the activity ofhBBP in eight mice in the hindquarter ectopic bone formation assaymethod using 5 μg rhBMP-2 alone (control) or 5 μg rhBMP-2 plus 0.05 mghuman BBP (hBBP). After 4 weeks, the animals were killed and thehindquarter removed. X-ray and DEXA analysis were conducted.

Results: hBBP was tested in combination with rhBMP-2.

When implanted, hBBP with BMP resulted in a greater amount ofcalcification induction than BMP alone.

TABLE 3 Densitometric quantitation of ectopic bone formation withvarious amounts of BBP implanted with 5 μg of rhBMP-2. Mean, SE (n).Group Mean BMC content (g) rhBMP-2 (5 μg) 0.00775 hBBP (0.05 mg) +rhBMP-2 (5 μg) 0.01125

1. An isolated peptide comprising the amino acid sequence of SEQ ID NO:11, or a fragment thereof, wherein said peptide or fragment increasesthe degree or rate of osteogenesis by BMP-2.
 2. The isolated peptide ofclaim 1, wherein said fragment increases the degree or rate ofcalcification in vertebrate cells.
 3. The isolated peptide of claim 1,wherein the amino acids represented as Xaa at positions 6, 9, 10, 11,13, 14 and 16 are selected from the amino acid group: K, R, H, Q, A, E,and W.
 4. The isolated peptide of claim 1, wherein the amino acidsrepresented as Xaa at positions 7, 17 and 18 are selected from the aminoacid group comprising: M, V, I, R, A or H.
 5. The isolated peptide ofclaim 1, wherein the amino acid represented as Xaa at position 6 isselected from the amino acid group consisting of: K, R, and Q.
 6. Theisolated peptide of claim 1, wherein the amino acid represented as Xaaat position 7 is selected from the amino acid group comprising: M, V orI.
 7. The isolated peptide of claim 1, wherein the amino acidrepresented as Xaa at position 9 is selected from the amino acid groupconsisting of: A, K and V.
 8. The isolated peptide of claim 1, whereinthe amino acid represented as Xaa at position 10 is selected from theamino acid group consisting of: E, Q and G.
 9. The isolated peptide ofclaim 1, wherein the amino acid represented as Xaa at position 11 isselected from the amino acid group consisting of: Q, K and R.
 10. Theisolated peptide of claim 1, wherein the amino acid represented as Xaaat position 13 is selected from the amino acid group consisting of: Qand K.
 11. The isolated peptide of claim 1, wherein the amino acidrepresented as Xaa at position 14 is selected from the amino acid groupconsisting of: N, G and D.
 12. The isolated peptide of claim 1, whereinthe amino acid represented as Xaa at position 16 is selected from theamino acid group consisting of: W and H.
 13. The isolated peptide ofclaim 1, wherein the amino acid represented as Xaa at position 17 isselected from the amino acid group comprising: A or V.
 14. The isolatedpeptide of claim 1, wherein the amino acid represented as Xaa atposition 18 is selected from the amino acid group comprising: R or H.15. A composition comprising an isolated peptide having the consensusamino acid sequence of SEQ ID NO: 11, or a fragment thereof, whereinsaid peptide or fragment increases the degree or rate of osteogenesis byBMP-2, and a molecule selected from the group comprising: TGF-beta,BMP-2, BMP-4, BMP-7 or demineralized bone matrix.
 16. The composition ofclaim 15, wherein the peptide comprises the amino acid sequence of SEQID NO: 13, or a fragment thereof, wherein said fragment increases thedegree or rate of osteogenesis by BMP-2.